🦠 Vibrio spp. in food
Detection of potentially enteropathogenic Vibrio spp. (V. parahaemolyticus, V. cholerae, V. vulnificus) in food by enrichment in ASPW and isolation on TCBS agar.
Overview
Vibrio is a genus of Gram-negative, halophilic (salt-loving) bacteria, naturally occurring in marine and estuarine waters. Three main enteropathogenic species are: V. parahaemolyticus — most common cause of bacterial food poisoning associated with seafood (diarrhea, cramps, fever, 12–24 h after consumption); V. cholerae — cholera causative agent (O1 and O139 strains) and milder gastroenteritis (non-O1/non-O139 strains); V. vulnificus — most dangerous species, causing septicemia with >50% mortality in persons with liver diseases.
The PN-EN ISO 21872-1 standard describes a horizontal method for detecting these three Vibrio species in food (mainly seafood: clams, oysters, shrimp, fish) and environmental samples. The method includes selective enrichment in alkaline saline peptone water (ASPW — Alkaline Saline Peptone Water), isolation on TCBS (Thiosulfate Citrate Bile Salts Sucrose Agar) agar and second selective medium, and biochemical and/or molecular (PCR) confirmation.
ASPW provides alkaline (pH 8.6) and saline (NaCl 2–3%) conditions favoring Vibrio growth, while inhibiting many other bacteria. TCBS agar is highly selective medium — bile salts and sucrose differentiate Vibrio species: V. parahaemolyticus (green-blue colonies, sucrose-negative), V. cholerae (yellow colonies, sucrose-positive), V. vulnificus (green colonies, sucrose-negative).
Vibrio testing is gaining importance due to climate warming — Vibrio are spreading to new water bodies (Baltic Sea, North Sea). In EU, testing is required as part of official control of seafood and shellfish.
Method principle
Sample is enriched in two sequential portions of ASPW (Alkaline Saline Peptone Water, pH 8.6, NaCl 2–3%). First enrichment stage: 41.5°C/6 h and/or 37°C/6 h. Second stage: transfer to fresh ASPW, 41.5°C/18 h (V. parahaemolyticus, V. cholerae) or 37°C/18 h (V. vulnificus). High pH and salinity of ASPW selectively promote Vibrio growth. After enrichment, plate on TCBS agar (37°C/24 h) and second medium. On TCBS V. parahaemolyticus forms green-blue colonies (sucrose −), V. cholerae — yellow (sucrose +), V. vulnificus — green (sucrose −). Confirmation: biochemical tests (oxidase +, growth with NaCl, Voges-Proskauer reaction) and/or PCR (toxin genes: tdh, trh for V. parahaemolyticus; ctx for V. cholerae).
Applications
- Seafood quality control — clams, oysters, mussels, shrimp
- Fish and fish products testing, especially raw (sushi, sashimi)
- Monitoring marine and estuarine water in shellfish harvesting areas
- Official control of imported seafood
- Epidemiological investigation in outbreaks after seafood consumption
- Climate monitoring — tracking Vibrio spreading to new water bodies (Baltic)
- Salads and dishes with seafood testing (ready-to-eat)
Key parameters
| Parameter | Value |
|---|---|
| Enrichment I | 41.5°C/6 h and/or 37°C/6 h in ASPW |
| Enrichment II | 41.5°C/18 h or 37°C/18 h in fresh ASPW |
| Isolation | 37°C/24 h on TCBS Agar |
| Isolation medium | TCBS Agar (Thiosulfate Citrate Bile Salts Sucrose) |
| Colony type | V. parahaemolyticus: green-blue; V. cholerae: yellow; V. vulnificus: green |
| Confirmation | Oxidase (+), biochemical tests and/or PCR (tdh, trh, ctx) |
Standard
- Standard number
- PN-EN ISO 21872-1:2017
- Title (PL)
- Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda oznaczania Vibrio spp. — Część 1: Wykrywanie potencjalnie enteropatogennych Vibrio parahaemolyticus, Vibrio cholerae i Vibrio vulnificus
- Title (EN)
- Microbiology of the food chain — Horizontal method for the determination of Vibrio spp. — Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus
Step-by-step procedure
1. Sample preparation
Weigh 25 g sample (seafood) into stomacher bag. Add 225 mL ASPW (pH 8.6). Homogenize for 60–120 s.
2. Enrichment — stage I
Incubate at 41.5 ± 1°C for 6 h (V. parahaemolyticus, V. cholerae). In parallel, you can incubate second portion at 37 ± 1°C for 6 h (V. vulnificus).
3. Enrichment — stage II
Transfer 1 mL from enriched culture to 10 mL fresh ASPW. Incubate 41.5°C/18 h (V. parahaemolyticus, V. cholerae) or 37°C/18 h (V. vulnificus).
4. Isolation on TCBS
Streak from enriched broth onto TCBS agar plate and second selective medium (e.g., chromogenic). Incubate 37 ± 1°C for 24 ± 2 h. NOTE: Do NOT autoclave TCBS agar — dissolve by boiling and pour into plates.
5. Colony reading on TCBS
Look for typical colonies: V. parahaemolyticus — green-blue, 2–3 mm; V. cholerae — yellow, smooth, 2–3 mm; V. vulnificus — green, 1–2 mm.
6. Subculture on non-selective medium
Select min. 3–5 typical colonies from each plate. Subculture on agar with NaCl (TSA + 1% NaCl). Incubate 37°C/24 h.
7. Confirmation tests
Perform: oxidase test (+), growth test with different NaCl concentrations (0%, 3%, 6%, 8%, 10%), sucrose fermentation test, Voges-Proskauer reaction, string test. Or confirm by PCR (genes: tlh/tdh/trh for V. parahaemolyticus; ctx/ompW for V. cholerae; vvhA for V. vulnificus).
8. Reporting
Result: "V. parahaemolyticus / V. cholerae / V. vulnificus detected/not detected in 25 g". State species and virulence factors profile (if PCR performed).
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Laboratory incubator 37°C and 41.5°C | Binder BD 56, Memmert INB 200, POL-EKO CLN 53 | 5,000–18,000 PLN |
| Class II laminar flow cabinet | Thermo Scientific MSC-Advantage, ESCO Airstream AC2 | 25,000–60,000 PLN |
| Stomacher / homogenizer | Seward Stomacher 400 Circulator | 12,000–25,000 PLN |
| Laboratory pH meter | Mettler Toledo SevenEasy, WTW inoLab pH 7310 | 3,000–10,000 PLN |
| Laboratory autoclave | Tuttnauer 2540M, Systec VX-65 | 15,000–45,000 PLN |
| PCR thermal cycler (for molecular confirmation) | Bio-Rad CFX96, Applied Biosystems 7500 | 40,000–120,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| ASPW (Alkaline Saline Peptone Water) | — | Peptone, NaCl 20–30 g/L, pH 8.6 ± 0.2; for selective Vibrio enrichment; 500 g package |
| TCBS Agar (Thiosulfate Citrate Bile Salts Sucrose) | — | Highly selective medium: sodium thiosulfate, sodium citrate, bile salts, sucrose, agar; 500 g package. Do not autoclave — only dissolve and pour! |
| Sucrose | 57-50-1 | TCBS component — differentiates Vibrio species (fermentation → yellow colonies) |
| Sodium thiosulfate | 7772-98-7 | TCBS component — indicator of H₂S production |
| Bile salts (Ox Bile) | — | Selective agent in TCBS inhibiting Gram-positive bacteria; 8 g/L |
| Sodium chloride (NaCl) | 7647-14-5 | ASPW and media component — Vibrio require NaCl for growth (halophilic); 20–30 g/L |
| Oxidase reagent | — | For confirmation test — Vibrio oxidase-positive; strips or drops |
| PCR kits (tdh, trh, ctx) | — | For molecular confirmation of virulence factors; optional |
Health and safety (OHS)
- V. cholerae — BSL-2 pathogen (non-O1/non-O139 strains) or BSL-3 (O1/O139), follow biological safety guidelines
- V. vulnificus — >50% mortality in persons with liver diseases, special caution with cuts
- Work exclusively in class II laminar flow cabinet
- Bile salts in TCBS — irritating, use gloves and goggles
- Autoclave used materials before disposal (121°C/20 min)
- Surface disinfection with 70% ethanol — Vibrio sensitive to desiccation